MetScape 3.1 User Manual
Contents
1. PIEAPONCIBI National Center for Integrative Biomedical Informatics MetScape 3 1 User Manual An App for Cytoscape National Center for Integrative Biomedical Informatics March 2015 2014 University of Michigan This work is Supported by the National Center for Integrative Biomedical Informatics through NIH Grant 1U54DA021519 01A1 and the Michigan Regional Comprehensive Metabolomics Research Core through NIH Grant U24DK097153 Contents OVER VIEN ec neta eet nee tT eer ee eee ee er ert er err eer ert er 4 POUT data SUC rset sa greece nung avec seca enero sed aceasta ese ns 4 Workflow ire 601 een neni a eee ene ee ee eer er ree 4 Installing Cytoscape and the MetScape app on your local computer cccccceeccseeeseeeeeseeeeeees 5 PART 1 PATHWAY BASED NETWORK ssssceeccccceccccceeeeaaaeessssseeseeeccececeeeeessssaaaaasssseseeeeeseseeeeseees 6 Enare Oe een eee E E E ee eee E ee eee ee 6 Option 1 Enter a list of COMPOUNGAS cccccceesccccceseccecescccceesececeeeececeeesecessuneceeeeneceesausecetseneceeseees 6 Option 2 Load an experiment file ec cccccccessececcessececeeseceeeeeecceeeeeecesseneceseegeceessueeessenecetegens 9 Pathway Based Visualization cccccccccccssscccceeseccceeseccceeeccceeusececcueeceeeeusecesauneceseeneceeseneecessunecesseges 13 PVCS CAPS TaD sco ect saree acest ces EEEE cesta vorentesneeenedeaeeeoace 14 Rules Used to B2. Control Panel fg Network style Select MetScape Build Pathway based Network Input Organism Human Data Files 4 Add Compounds Compounds none Genes rone Compounds Input ID Input Name DL Lipoyl L lysine trimethylalamine e glucose Genes i ar Input ID Input Symbol Query Use selected pathway TCA cycle Node Table Edge Table Network Page 6 of 39 3 Select a species Choices are human rat mouse 4 Manually enter or copy and paste compound ID s or name s and or Entrez gene ID s or symbol s Click Add and enter the appropriate ID type in the popup box Lists of IDs should be separated by spaces lists of compound names should be entered one per line 5 Click OK 6 If you enter a compound name it will map to its KEGG ID a popup window will appear If there is more than one potential match use the dropdown arrow to choose the best match If the compound is not found in the system it will say Not Found The mapping selection will be saved so that your selection will appear as the default option in the future Select names for compounds One or more of fle compounds in your query had multinle matches in the datahase Please select the mato ihat i best or none if you do not wish the compound fo aopear in fhe results Input Name Potential Matches DL Lipoyl _tysine DL Lipoyl _tysine a 7 Click OK 8
3. 0 158654 1 0 130999 0 130999 0 08656 0 12006 0 09012 0 164396 0 441288 0 071866 0 282938 0 417381 0 322295 0 266963 0 004821 0 104243 0 02707 0 156343 0 450516 0 21722 0 08650 0 417381 1 0 773163 0 775301 0 131547 0 046118 0 517814 0 01262 Page 33 of 39 The first row and column contain metabolite names Below is a portion of an example matrix format correlation file G Leucine 0 287503 0 106856 0 12006 0 322295 0 773163 1 0 884062 0 165156 0 023038 0 420597 0 106846 The rest of the rows columns contain correlation values H Isoleucine Threonine 0 352786 0 175509 0 09012 0 266963 0 775301 0 864062 1 0 258082 0 09493 0 452117 0 066391 0 213065 0 074988 0 164396 0 004621 0 131847 0 165156 0 258682 1 0 553733 0 306851 0 002028 Building a Network 1 To begin a Cytoscape session with the MetScape app first start Cytoscape 2 Select Apps gt MetScape gt Build Network gt Correlation based from the Cytoscape menu 3 A MetScape tab now appears on the left of the Cytoscape screen 4 Use the Select button to upload the appropriate file 5 Compound names will map to their KEGG id a popup window will appear If there is more than one potential match use the dropdown arrow to choose the best match If a compound is not found in the system it will say Not Found The mapping selection will be saved so that your selection will appear as the default opt
4. SUPPLEMENT METDISEASE APP MetDisease is designed to annotate metabolites with Medical Subject Headings MeSH disease terms The underlying data comes from the Metab2MeSH data set Sartor et al Bioinformatics 2012 May 15 28 10 1408 10 To use MetDisease you need a network of compounds with KEGG or PubChem Ids or compound name If using MetScape 2 3 2 or higher you can use MetDisease on the MetScape networks How to use MetDisease Once you have built a MetScape network Page 38 of 39 1 Select Apps gt MetDisease gt Find MeSH Terms 2 Choose the appropriate identifier type and attribute Click OK 3 A hierarchical tree of MeSH Disease terms should be displayed in the Table Panel 4 Selecting a tree node will select respective metabolites in the network The numbers shown after the descriptors represent the number of matching metabolites in the active network Note Descriptors that have no nodes in the active network can be collapsed and hidden 5 To view relevant PubMed citations right click on a selected compound node and select MetDisease gt PubMed Citations For more information about MetDisease go to http metdisease ncibi org The MetDisease user manual can be downloaded at http metdisease ncibi org pdf MetDisease User Manual pdf Page 39 of 39
5. compounds or genes When any other Page 14 of 39 Network Type is selected the only option is to use compounds genes unless using a selected pathway e Add or Remove data e Select a pathway o Use to view all the compounds and reactions associated with a metabolic pathway Select a specific pathway from the drop down list Saturated fatty acids beta oxidation Selenoamino acid metabolism e Save the Output as a File e Build Network graph based on the data Control Panel Bg Network style se Build Pathway based 7 Network Input Organism Human Data Files Add Jf Remove Clear Reset _ if Input ID i Input Symbol Add or remove ACAT1 A ACLY I L NOOSE Options Network Type Compound Query Use compounds v D Use selected pathway TCA cyde puid Network Page 15 of 39 Note When the organism is human the Genes section will show Input ID and Input Symbol However when the organism is not human ex rat the Genes section will Show Input ID Input Symbol and Human Symbol Rules Used to Build Different Network Types e Compound Reaction Enzyme Gene C R E G Compound Reaction C R and Compound Gene C G networks The C R E G C R and C G networks are all built from the same underlying data That data is derived in each case by finding compounds that participate in reactions that are catalyzed by enzymes that are encoded by genes If
6. 2 4 73898475 0 00822755 0 0447139 up _ 24368 24401 25721 81683 Antigen processi KEGG Pathway 21 0 2496329 4 71793226 0 00841626 0 0447139 up 24223 24812 25217 25599 1 2 3 A 5 6 T 9 To generate a concept file you can click the LRpath button from MetScape that will take you to the LRpath website Note LRpath performs gene set enrichment testing an approach used to test for predefined biologically relevant gene sets that contain more significant genes from an experimental dataset than expected by chance Sartor et al 2009 To run LRpath you need a Gene Expression file with fold change or log fold change values and p values The Gene Expression file needs to contain all gene records not just those for significant genes LRpath will determine the significant genes from the input Example of selecting experimental data including importing files and designating fold change p values and thresholds Select Experimental Data Choose species Data Organism Human P value Fold Change Compounds and Threshold Select experimental data none Import File P value none hreshold Fold Change none Import File Genes button Select experimental data none P value none Fold Change none Concepts Select experimental data none v Import File Generate a concept file from genes using LRPath l LRXPath v r Button for accessing l OK Il Cancel
7. 8 25757 140547 Alanine andaspaKEGG Pathway 1s 0 47651019 19 323541 1 93E 06 8 75E 05 up 24379 24401 25721 81670 81829 Reductive carbo 24368 24399 24401 25721 79250 Oxidative phosp KEGG Pathway 44 0 27758123 5 61284966 3 64E 05 9 89E 04up 116550 291103 295923 301011 311 Urea cycle and up 24368 24379 24399 24401 24600 PPAR signaling p KEGG Pathway 32 0 28585824 5 90912002 _2 33E 04 0 00441518 up 24158 24450 25045 25757 29171 Carbon fixation 0 44333373 15 7233268 3 59E 04 0 00542234 up 24401 25721 81670 81829 114508 Arginine and pro 24368 24379 24399 24401 24600 Butanoate metal KEGG Pathway 30 0 26301141 5 12695967 0 00101503 0 01254945 up 24379 24399 24401 24450 25721 Valine leucine aKEGG Pathway 24 0 27898384 5 66198894 0 00167291 0 01625109 up 24158 24450 29711 140547 17046 Glutathione met KEGG Pathway 24 0 27372392 5 4799003 0 00205204 0 01860519 up 24379 24399 24401 24492 25721 Propanoatemet KEGG Pathway 16 0 32105291 7 35380592 0 00265316 0 02122524 up _ _ 24158 140547 170465 171155 298 Nitrogen metabqKEGG Pathway 15 0 30373897 6 60362465 0 00596513 0 04056287 up 24379 24399 24401 25721 29242 6 C5 Branched dib up 24158 24379 24399 24401 25721 Pyruvate metabd KEGG Pathway 19 0 26689922 5 25234187 0 00714807 0 04226683 up _ 1829 298942 306198 307858 361 Phenylalanine mKEGG Pathway 21 0 2503493
8. After the experimental data has been loaded if any of the genes compounds that you submitted were not mapped to the database objects a Missing Data window will appear After viewing the Missing Data information click SAVE to save the data for later viewing or click OK to close the window without making it available for future viewing See the Missing Data Window section below under Option 2 Load an experiment file for more information about missing data Page 7 of 39 The following input data could not be found in the MetScape database 9 Select a Network Type by selecting one of the following from the dropdown menu e Compound Reaction Enzyme Gene 1 e Compound Reaction 2 e Compound Gene 3 e Compound 4 Note When selecting Compound as the Network Type a dropdown menu appears under Query providing the option of choosing between compounds or genes When any other Network Type is selected the only option is to use compounds genes unless using a selected pathway 10 Click Build Network to query the database and create the network Page 8 of 39 Option 2 Load an experiment file Use to load experimental data to visualize and explore compound networks over time or in varying experimental conditions The input can be an Excel comma or tab delimited file MetScape allows users to load three types of files compound file gene file and concept file Each type is optional e g you can load only compounds only g
9. Data window will appear Genes compounds and concepts may appear on this missing elements list because Page 12 of 39 e Genes and compounds that you supply may not be in the database If they are not found in the database then they are reported as missing e If your input genes are not human Rat for example then they are mapped to human genes using homologs from NCBI s HomoloGene If this mapping fails then those genes are reported as missing e MetScape will display only the genes that encode metabolic enzymes If an input gene does not encode metabolic enzymes it will appear on the missing elements list e A concept pathway will appear on the missing list if all of its significant genes are missing The list of significant genes for a concept comes from the input file or from LRpath After viewing the Missing Data information click SAVE to save the data for later viewing or click OK to close the window without making it available for future viewing Missing Data Why are these elements missing The following input data could not be found in the d MetScape database Compounds Creatne Crn GSH Lysine Lysine Arginine OH Butyrate PtdCholine PtdEthanolamine TMAO Betaine Total Glucose Total Glutathione Pathway Based Visualization MetScape includes a legend explaining its various shapes and colors The legend will be specific to the current network type pathway or correlation Access th
10. LRpath website Page 11 of 39 Below is an example of the ID mapping window from a loaded compound experiment file The mapping selection will be saved so that your selection will appear as the default option in the future Select Compounc Select names for compounds One or more of the compounds in your query had multiple matohes in the da Please select the maich thats best or none if you do not wish the compound fo appear in the results Input Name Protein glutamine Protein L glutamine Protein M5 alkylglutamine none GIL ln LEUL IE lot Found NotFound Notpound Noto Notpound Sphingomyelin Sphingomyelin Notpond Notpound otrond Notpound o E er legai ooo O Note If you select Save on the Select Compound Mappings window you will get a csv file containing mapping information for all compounds that MetScape successfully mapped Note Multiple networks can be built within the same user session After a network is built pull up the Select Experimental Data window by clicking on the Select button and change the data files Then build the new network Both networks will remain accessible If you change species however all existing data will be lost The below warning window will pop up in this situation Missing Data Window After the experimental data has been loaded if any of the genes compounds that you Submitted were not mapped to the database objects a Missing
11. MN database Pathway specific Compound networks are built similarly to the Compound networks built from a set of input genes Table Panel Display Attributes of compounds reactions pathways and concepts that you have selected in the network appear in the Cytoscape Table Panel Choosing attributes provided through MetScape Page 16 of 39 To choose which attributes to view 1 To choose attributes for compounds click the Node Table tab in the Table Panel You can choose to view node edge or network attributes by clicking on the respective tabs at the bottom of the Table Panel Pathway data can be viewed under the Pathway Filter tab and concept data can be viewed under the Concept Filter tab in the Table Panel 2 Click the Hide All Columns icon in the Table Panel toolbar This clears the Table Panel of all columns 3 Click the Show Column icon in the Table Panel toolbar A list of attributes with check boxes will appear Table Panel D ses oo m amp f z Network Show Column icon Hide All Columns icon Node Table Edge Table Network Table Pathway Filter 4 Select the attributes you want displayed in the Table Panel Attributes are listed with item type before the attribute ex Compound name Enzyme name The available attributes will depend on the type of network created Table Panel g se o 5 wW f z V Compound name a V Enzyme name 4 5 When you are done click anywhere outside o
12. The concept information can be saved as a file To save this information 1 Under Concept Filter tab click the Save Concepts button 2 Anew window pops up asking where to save the file Choose the desired location 3 Click Save The file saves as a CSV file that can be opened with most text editing or spreadsheet programs The file format is identical to the input concept file lotrate c cycle TCA cyde Cytokine cytokine receptor miersii Page 23 of 39 MetScape Results Panel Additional Node Information Additional information can be obtained for each node by double clicking on it To get additional information about any compound gene reaction or enzyme 1 Double click on the node of interest 2 On the right side of the screen the MetScape Results Panel will show up Entrez Gene ID Human 2572 glutamate decarboxylase 2 pancreatic islets and brain 65kDa Subcellular location s Golgi apparatus synaptic vesicle membrane perinuclear region of cytoplasm axon cytoplasmic membrane bounded vesicle Animation of Data Create an animation of the data to see how it changes over time and across treatments Building an Animation 1 Create a subnetwork for example create a subnetwork for the TCA Cycle See the Concept Filter section in the Table Panel section for how to do this Note Itis not necessary to create a subnetwork before doing dat
13. W OU rosie ccc ctehee seecncdcacetcpaciencweassecediesnanxnsdanneeesksndirasuanasuendeusareuchabatasehcoayuentesnieeseuearacc 29 Option 1 Expanding a network in the current network window Example using Compound Reaction Network 5c 5ccescsc aca ssc peta eaters cto ot sta wes ean ema eae denn naseee er cecnne notes 29 Option 2 Expanding a network in a new window Example using Compound Network from Sele EEGEN Jaane ne ee er E eee eee 30 Collapsing a MOTWOIK scccsciscccasaecscersaivncisageceseinsdeanteriacsgesoesenadnbitescadearasudesauiadedaaavsedesntasstenaneseuainee detainees 31 Creating a subnetwork oiticencssacccedsdedesoincecesnictecesticscn ndidedetniedsainededadanis dsaasiwasgadesoaaataaneasieediasee oleate 31 Destroying a W011 eG Cee nee eee eee 32 PART 2 CORRELATION BASED NETWORK ccccccccccccccccccceseeesssseseeeeecccceeeceessssauaaeassssseeeeeseeeeeeeees 33 Page 2 of 39 Entering Data ciiciiees ee a es ie ee N EE EO 33 FIE OGY ACS cep dics ic ost ceca ar csiv ns dasnscet ap anise N 33 B ildinge a NCTWORK i565 toss tenrapectdncae tics a a aidan Moats oda E S 34 Group Denton eci O wsesardstenthin se euendasaiels wakes andes case uses os 36 Correlation Vis alizatiO Mna a E E O E RS 36 Additional Informati n neemen A E A R Rr Ei 37 Correlation Network Results Panel ccccccccccccccsasssseeccccceeaeeeseeccceeessueeseeecceeessuuaaseeeceeeesaaaaasses 37 SAVING AND REOPENING A SESSION 002 5 cccicciindec
14. a animation the animation can be done on any network For example the Compounds file can have additional data columns see image below that are loaded when initially importing the Compounds file Page 24 of 39 A CID C00219 CO0503 CO018 CO2 21 C0004 C1686868 C05122 CO0064 C05629 COO0079 C00398 C00294 B p value 0 040982 0 008525 0 030047 0 024311 0 017356 0 004029 0 000312 0 000021 0 000252 0 036563 0 016353 0 000014 C Fold change 1 49 1 53 1 35 2 01 1 03 2 01 1 75 1 2 1 38 1 15 1 07 1 4 D t10 0 477255 0 902665 0 489314 0 80923 0 312654 0 19994 0 002804 0 113069 0 775584 0 139549 0 115421 0 125432 E t11 0 811411 0 098903 0 029276 0 125058 0 651588 0 379436 0 47 7482 0 635171 0 1984 74 0 464563 0 541825 0 275978 F t1 2 0 907349 0 509585 0 31459 0 843886 0 366625 0 374019 0 460432 0 490499 0 03 7484 0 8363 0 741933 G t2_ 0 677623 0 33507 0 703353 0 667656 0 413499 0 004547 0 683021 0 769097 0 713089 0 041068 0 829824 0 513364 H t21 0 349178 0 761891 0 0648 72 0 008175 0 650567 0 325439 0 562158 0 756346 0 597104 0 001483 l t2 2 0 686385 0 042653 0 340032 0 131301 0 926665 0 605136 0 268456 0 783052 0 96571 0 616224 0 302741 0 575744 2 From the Apps menu choose MetScape gt Animate Tools Help App Manager Agilent Literature Search GeneMANIA MCODE MetDisease MetScape Reactome FI Ani
15. ccepted column based is recommended 1 Column based e The first row must contain column headings of the user s choosing e The first two columns contain metabolite names e The next column s contains values such as p values Below is a portion of an example column based correlation file A metabl Alanine Alanine Alanine Alanine Alanine Alanine Alanine Alanine Alanine Alanine Alanine Alanine Alanine Alanine B metab Alanine Sarcosine Glycine Alpha am Valine Leucine Isoleucine Threonine Serine Proline Asparagin Aspartic a Methionir 4 Hydroxy c pcor 1 0 110571 0 255278 0 091832 0 040047 0 006732 0 071432 0 100668 0 13329 0 468989 0 05741 0 059375 0 102612 0 11061 2 Matrix format A Alanine sarcosine Glycine Alpha am Valine Leucine Isoleucine Threonine Serine Proline B C D pval 0 0 101242 0 000101 0 1743 0 554916 0 921006 0 291444 0 136092 0 047578 3 19E 15 0 396934 0 360926 0 128614 0 101123 D E qval 0 0 287501 0 001017 0 428134 0 74299 0 960585 0 548247 0 358658 0 164594 6 56E 13 0 616099 0 609963 0 346165 0 287501 E F Alanine Sarcosine Glycine Alpha ami Valine 1 0 215426 0 215426 0 171592 0 167991 0 450516 0 287503 0 352786 0 213065 0 070274 0 593237 0 110042 ine 0 171592 1 0 000618 0 060618 0 158684 0 21722 0 106856 0 175509 0 074988 0 03839 0 129416 0 039533 0 167991
16. e MetScape Legend from Apps gt MetScape gt Show Legend Page 13 of 39 Legend Compound C Reaction Enzyme C Gene C Input B O Significant C O Up regulated gt O Down regulated Co O Expansion EE Close Additional information about the network is expressed through visualization e A compound is red in the original network and subsequent subnetworks if it was in the original data loaded into MetScape e A green border surrounding a node represents a significant gene compound e Node size represents the direction of the change Larger nodes represent an increase and smaller nodes represent a decrease in gene metabolite The actual amount of the change is not represented visually e When a node is expanded the edges between the original node and the expanded nodes become blue Note In a pathway based network selected edges are highlighted yellow instead of red MetScape Tab This tab has options for choosing Network Types the data you entered and more From this tab you can e Select between building a pathway based network or a correlation based network e Select the type of network that you want to build Select Network Type by selecting one of the following from the dropdown menu o Compound Reaction Enzyme Gene o Compound Reaction o Compound Gene o Compound Note When selecting Compound as the Network Type a dropdown menu appears under Query providing the option of choosing between
17. emaining columns contain experimental data Multiple experimental values are permissible in the same spreadsheet Below is a portion of an example gene file with Gene IDs significance values and fold change values A B C 1 ENTREZ GENE ID Pvalue Log_fold_change 2 1 0 002335904 0 02 3 2 2 07E 08 0 06 4 2 2 03E 08 0 04 a 9 0 003915472 0 04 6 T G 9 10 0 00026716 0 08 12 0 000237925 0 05 13 0 002489364 0 05 14 0 506396205 0 00 15 8 25E 06 0 04 16 0 022222108 0 01 1 1 47E 08 0 07 1 2 24E 06 0 09 1 2 12E 05 0 07 19 0 820594937 0 00 19 1 76E 08 0 06 19 9 46E 09 0 04 19 0 660631201 0 00 20 1 16E 07 0 05 Concept File The concept file can be generated by a gene set enrichment analysis tool such as LRpath or GSEA from gene expression data Note Gene set enrichment testing is an approach used to test for predefined biologically relevant gene sets that contain more significant genes from an experimental dataset than expected by chance e GSEA Subramanian at al Proc Natl Acad Sci USA 2005 102 15545 15550 e LRpath Sartor et al Bioinformatics 2009 25 2 211 7 Below is a portion of an example concept file Page 10 of 39 A B G D E F G H I Concept name ConceptType n genes coeff odds ratio p value FDR Direction sig genes Citrate cycle TCAKEGG Pathway 32 0 44858373 16 2447876 1 23E 08 _1 67E O6lup _ 24368 24399 24401 25179 25721 Fatty acid metab up 24158 25363 2561
18. enes only concepts or any combination of the above 1 Select Apps gt MetScape gt Build Network gt Pathway based from the Cytoscape menu 2 A MetScape tab now appears on the left of the Cytoscape screen 3 Select a species Choices are human rat mouse 4 Load experimental data by clicking the Select button on the MetScape tab Compound File The compound data file must meet the following requirements e The first row must be a heading row in which o The columns in the first row are column headings to label the data e All other rows contain experimental data in which o The first column contains KEGG Compound IDs or names o The remaining columns contain experimental data Multiple experimental values are permissible in the same spreadsheet Below is a portion of an example compound file with Compound IDs significance values and fold change values B C Fold Change P value 0 51 0 006174265 114 0 645801 57 0 99 0 9 70303452 2 12 0 0660526 75 0 91 0 11537626 7 0 99 0 904658012 0 95 0 5 4835153 1 09 0 8091 72269 0 56 0 100 24407 1 36 0 1899 9302 0 92 0 755789133 0 46 0 166 0 7182 1 22 8 0 805151078 0 60 0 356405722 1 2 3 e 4 i 6 i amp 9 Page 9 of 39 Gene File The gene file must meet the following requirements e The first row must be a heading row that includes column headings to label the data e The first column contains Entrez Gene IDs or Official Gene Symbols e The r
19. f the list and the table will be populated with the appropriate data Note In Cytoscape 3 0 clicking on the two horizontal squares with check marks will select all attributes Below is a table of available attributes for each node type Page 17 of 39 Attribute ID Compound approximatemMw Compound casnum Compound cid Compound file_name Compound formula Compound mw Compound name Compound pubchemcid Compound smiles Compound synonyms Enzyme ecnum Enzyme name Gene file_name Gene column_name Gene description Gene human geneid Gene locations Gene symbo l Gene geneid Reaction equation Reaction locations Reaction pathway Reaction reversible Reaction rid Type canonicalName a 7 Description Unique identifier required by Cytoscape Compound Molecular weight integer data source EHMN database Chemical Abtracts Service compound identifier KEGG compound unique identifier Rows with data specific to your experiments Molecular formula Molecular weight Compound name PubChem compound identifier SMILES string for compound Other names for compound Enzyme Comission number Enzyme name Relative gene data values Gene data p value Gene description Human gene identifier Subcellular location Official gene symbol Ortholog gene 10 if the input organism is not human Equation for a reaction Reaction subcellular location EHMN database Pathway involving reaction T reaction is reversible F reacti
20. graph click on the concept name in the Table Panel The concept will then be highlighted in the graph associated nodes and edges turn yellow False Descoery Rate 0 104 Cytokine cytokine receptor interaction Dit eer eel i ere he raher Lia i Node Table Edge Table Network Table WordCloud Display Pathway Fiter Concept Fiter A subgraph can be created for a given concept The subgraph consists of genes considered to be significant to the concept those genes that drive the concept not all genes in the concept To create a subgraph 1 Select a concept in the Concept Filter tab 2 Click the Create Subnetwork button at the top of the Table Panel make sure you are on the Concept Filter tab 3 A new graph appears in the graph window This is the subnetwork for the selected concept Page 22 of 39 lt ss Compound Reaction Enzyn Compound Reaction EnS62 751 Compound Reaction En 26 1 0 369 0 Table Panel Seca caer Dace Al Corap Grene Sein Save Concepts Creamy Seen Cas Ember of Enriched Driving Genes Number of Genes in Network ft P value False Discovery Rate j 1 and 2 Meth 3 CHloroaaryic Acute myeloid lAdherens juncti Alanine and asg te Concept Filter The Reapply Selection button will re select the last selected concept in the graph If you select a concept and then select off of it clicking the Reapply Selection button will re select that concept
21. ion in the future Select Compound Mappings ne or more af fhe compound s in your query Aad multine matotes in fe datahase Please select the matoh that is best ar Ynane if you do not wish fhe compound fo appear in the results Potential Matches Jot Found jerand ae Desuomessd 00217 Pauanne coosio cere 00037 i 243 Carboxy 3 aminopr CO4441 lee 5 Click OK 6 Under Edge Mapping use the dropdown menu next to Base Edges on and select the appropriate column from your data file Note The Range for Edges slider changes to match the data type chosen In the example files used in this manual the slider is O to 1 for pval but 1 to 1 for pcor Range for Edges seen see Range for Edges J Negative Page 34 of 39 7 Under Edge Mapping use the dropdown menu next to Tooltip Labels to select additional values that will be viewable when mousing over an edge in the built network 8 Optional Load group definition file For more information go to the Group Definition section of this document 9 Under Range for Edges drag the arrows left and right OR enter numbers in the text boxes to select the desired significance range A log scale is used to allow for very small p values 10 The number of edges that will appear in the network built with the current parameters will appear below the slider Control Panel Ox MetScape fg Network style select wordcloua sets MetSca
22. kntilndticcniie tke tiin th Reacts 38 Savne a SESSION asics sere tos ooee wah eet seats Reece tae sao hewn Seca needa acest oes he eee 38 REODENING a SesSsSlOMoc sel ie eece Shed cate sel iets Leet etevte ecole A 38 SUPPLEMENT METDISEASE APP inai R E a E E lodeownes 38 HOW 0 USC VICEDISCASC en a a beliecatotnawestaehaeseecteceee 38 Please note that due to continuous software upgrades the images in this handout may not exactly mimic what you see on the screen Page 3 of 39 OVERVIEW About data sources MetScape is an app for Cytoscape the bioinformatics network visualization tool The app can be used to visualize and interpret metabolomics and gene expression data in the context of human metabolic networks MetScape uses a metabolite database developed by extracting and integrating information from the following sources 1 Edinburgh Human Metabolic Network EHMN http www ehmn bioinformatics ed ac uk 2 KEGG COMPOUND Database http www genome p kegg compound MetScape allows users to load a list of metabolites with experimentally determined concentrations a list of genes with experimentally determined expression values and a list of concepts or pathways and display them in the context of relevant metabolic networks Workflow overview With MetScape you can Trace the connections between metabolites and genes Integrate multidimensional data Visualize compound reaction enzyme and gene networks a
23. led start the application 3 To install the MetScape app select Apps gt App Manager from the Cytoscape menu 4 Under the Install Apps tab scroll down until you find MetScape in the second column s Install Apps Currently Installed Check for Updates Download Site http apps cytoscape org aa Search 3 0 2 installed 3 0 1 Maps human mouse and rat metabolomics and gene expression data to human metabolic networks and enables pathway analysis Install from File View on App Store Insta 5 Click on the MetScape app 6 Click Install 7 When installation is complete click Close A MetScape option is added to the Cytoscape Apps menu Page 5 of 39 Registration MetScape is a free program We ask you to register because it helps us to keep track of the number of downloads Your information will be stored in a secure database and we will not share it with anyone We may send you infrequent e mails about future MetScape releases Note This app requires Cytoscape 3 0 to run correctly PART 1 PATHWAY BASED NETWORK Entering Data 1 To begin a Cytoscape session with the MetScape app first start Cytoscape 2 Choose one of the following methods to get started Option 1 Enter a list of compounds 1 Select Apps gt MetScape gt Build Network gt Pathway based from the Cytoscape menu 2 A MetScape tab now appears on the left of the Cytoscape screen
24. mate BINGO Show Legend Filter By About MetScape Settings 3 A new window pops up titled I nitialize Coupled Animation of Multiple Data Columns 4 Column headings from your data are listed on the right side of the new window Determine how many animations number of rows and data items per animation number of columns you will need Enter this information into the Rows and Cols boxes If you change these numbers from the default click on Reconfigure Layout Page 25 of 39 amp gure Layout Column 1 Undefined Drag and drop labels from the ist on the night into the table of data labels for each animation Set the number of Drag and drop labels from the ist on the right into the table of data labels for each animation Set the number of animations number of rows and the number of data items per animation number of columns in the text felds and animations number of rows and the number of data items per animation number of columns in the text fields and DataSeries_0 t1_0 DataSeries_1 Undefined 6 After grid is completely filled in click on Build Animation Note If you want to undo what you filled out in the grid click on Restart and the grid will be cleared 7 Two new windows appear in the graph panel each representing a different treatment In addition an Animation Controls window appears Page 26 of 39 Animation Controls Netw
25. nd display compound structures as well as information for reactions enzymes genes and pathways Visually animate changes in compound concentrations over time and across experimental conditions Pathway Based Workflow The basic steps in the pathway workflow include 1 Enter data You can type or paste a list of compounds and or genes load a file containing experimental data or start from a biological pathway Select compound and reaction attributes Choose which attributes to display ina table as you work with your visual network graph Explore the visual network and table of attributes e Expand and collapse a network e Create a subnetwork e Visualize your data in a wide variety of network layouts provided by Cytoscape e Use color size and other effects to visually reflect a set of attribute values Save your session and reopen it later Page 4 of 39 Correlation Based Workflow The basic steps in the correlation workflow include 1 Enter data You can load a file containing experimental data 2 Select the desired Edge Mapping and Significance Range Use the slider to designate the desired significance range 3 Explore the visual network and table attributes 4 Save your session and reopen it later Installing Cytoscape and the MetScape app on your local computer l Install Cytoscape on your computer For more information go to the Cytoscape website at http cytoscape org 2 After Cytoscape is instal
26. on Page 19 of 39 compound pubchem 5280749 C05952 Leukotriene E4 p 6435286 _C05951 Leukotriene D4 XY gicolihochotate 7 5280749 C05952 6435286 C05951 de N Node Table Edge Table Network Table Pathway Filter To sort by an attribute click the attribute column heading to sort in the reverse direction click again Compound pubchemcid A A Compomdname al aU 7 5280749 C05952 6435286 A ceukotienebs C05951 o aa giycolthocholat CE5560 roxy 5beta cholan C03990 ANM beden ts OAAR Pathway filter in the table panel MetScape provides two ways to access pathway information The Pathway Filter tab lists all pathways represented in the network Selecting one or more pathways will highlight in the network all nodes in the pathway s Pathways are also displayed as attributes in the Node Table tab Page 20 of 39 Table Panel E a Pathways 3 oxo 10R octadecatrienoate beta oxidation Aminasugars metabolism Androgen and estrogen biosynthesis and metabotsm De novo fatty acid biosynthesis Dimethyl branched chain fatty acid mitochondrial beta oxidation Di unsaturated fatty add beta oxidation Node Table Edge Table Network Table Concent Fiter A subgraph can be created for a given pathway The subgraph consists of a subset of nodes from an active network that belong to the selected pathwa
27. on is non reversible Reaction identifier Node type gene enzyme reaction or compound Node name that is shown on node label Please note that the reactions shown in the Reaction equation attribute display all compounds while the networks built from the list of input genes compounds show only main compounds see Ma et al Mol Syst Biol 2007 3 135 for details Pathway specific networks show all compounds Below is a table of available attributes for each edge Attribute Enzyme names Enzyme ecnums Reaction equation Reaction locations Y Description Enzyme name Enzyme Comission number Equation for reaction Reaction subcellular location EHMN database Pathway involving reaction T reaction is reversible F reaction is non reversible Unique identifier for reaction Common name Direct or undirected interaction Type of interaction Reaction pathway Reaction reversible Reaction rid canonicalName direction 6 Select nodes and or edges in the graph to view their attributes in the Table Panel If nothing is selected attributes for all nodes and edges are shown Page 18 of 39 Notes e Some attributes such as Formula Mass and Smile apply only to compound nodes while others such as Enzyme Pathway and Reversibility apply only to reaction nodes Rearranging attributes in the table panel To reorder attributes in the Table Panel click on an attribute column heading and drag it to a new locati
28. only genes are input then all the enzymes reactions and compounds that match those genes are used If only compounds are input then all the reactions enzymes and genes that match those compounds are used If both genes and compounds are input then only those C R E G couplings that match both a compound from the compound input and a gene from the gene input are used If a concept file is provided genes from that file will be used as input instead of the genes from a provided gene file If only a gene file is provided all genes from that file are used as input In this case we recommend that you load a smaller set of genes e g most significant differentially expressed genes e Compound C networks built from compound input If a list of compounds Is provided the resulting network will include the query compounds shown in red plus any compounds that participate in the same reactions as query compounds The edges will be drawn between seed compounds and their neighboring compounds e Compound C networks built from gene input If a list of genes is provided the resulting network will include the compounds that are related to the query genes via the reactions in which they participate and the enzymes that catalyze these reactions The edges will be drawn between all compounds e Pathway networks Pathway specific C R E G C R and C G networks are built from a set of genes enzymes reactions and compounds defined in the EH
29. ork Edit 4 Select Group 4 Nested Networks a PN MetDisease MetScape p Cragg Subnetwork Expand Collapse Restore Original Network Expand in Existing Network Expand in Subnetwork External Links Preferences 3 Anew network Is created in a new window This network includes only the expansion seed compound and its related compounds reactions enzymes and genes depending on the Network Type The original color designations are used when the graph is created in a new window a Dihe Page 30 of 39 Collapsing a network To collapse a network that is expanded in a current network window 1 Zz Right click on the expansion seed compound node Select MetScape gt Collapse and then e To collapse only the branch expanded from that expansion seed compound select Collapse e To collapse all expanded branches select Restore Original Network Creating a subnetwork A subnetwork of a current network can be created and will appear in a separate window A subnetwork will include all highlighted nodes and edges selected nodes and edges Should be yellow For example 1 2 Select a compound in the built network For this example will select Sarcosine Go to the Select menu in Cytoscape Choose Nodes gt First Neighbors of Selected Nodes gt Undirected Now all the first neighbors of Sarcosine should be yellow Go to the Select menu in Cytoscape Cho
30. ork Compound Reaction Enzyme Gene Measurement Sets pS e mete x sg PPerats 7 Manipulating the animation zooming 1 Zoom in on one graph to the appropriate view size zoom the same way you do with any Cytoscape graph 2 On the Animation Controls window click on Realign All This brings all the treatment graphs to the same view amp Animation Controls Network Compound Reaction Enzyme Gene Measurement Sets gt lysine poe hydrogenase e TrormingTr Page 27 of 39 Manipulating the animation bar chart 1 The bar chart in the Animation Controls A window shows the range of the data and its 4 0 frequency The range of measurements are on the x axis while the frequency with which they occur in the experimental data set are on the 1 5 B 1 0 y aXIsS 0 5 0 0 0 0 0 2 0 4 0 6 0 8 1 0 2 Click on a bar in the bar chart and a vertical line slider will show up on the bar chart The slider can be moved by clicking on the bar chart and dragging the line When moved the values shown in black are also shown in black in the graph and the set of the color scale is reset 3 The selected portion of the graph determines what the animation will show This allows outliers to be removed if desired 4 The color range can be changed by clicking the dropdown arrow next to Blue Red Playing the animation To play the animation click Play on the Animation Controls window Color
31. ose Edges gt Select Adjacent Edges Now all the adjacent edges should be yellow Right click on Sarcosine A menu of options will pop up Pidencefom Literature Select Group ff Nester etworks Me Create Subnetwork MetScape Expand Reactome FI Collapse External Links Restore Original Network Preferences x AN AC ONT OTC RU r la all F Page 31 of 39 7 Choose MetScape gt Create Subnetwork Note When choosing options from the menu be careful not to move the cursor outside the menu panels Doing so will remove the highlighting of the nodes and edges resulting in an empty subnetwork 8 A new subnetwork is created in the graph window Subnetwork Destroying a network To destroy a network no longer needed 1 Make sure you really do want to destroy the network e Cytoscape will ask you to confirm the deletion e Destroying the network is irreversible e Note If you want to destroy a network view without destroying the network itself use Destroy View instead of Destroy Network 2 On the Network tab in the Cytoscape Control Panel right click on the network you want to destroy and select Destroy Network Note If you destroy a network that has subnetworks the subnetworks are NOT destroyed They are promoted up one network level Page 32 of 39 PART 2 CORRELATION BASED NETWORK Entering Data File Formats Two correlation file formats are a
32. pe Input Correlation Data Correlation File csv xdsx or x s smail data output pcor unknowns ki Edge Mapping Base Edges on pval Tooltip Labels qval Group Definition File csv xlsx or xds iem v _seect Range for Edges 3 E 9 1 00 Show Top 495 Edges by Count Edges 495 of 509 providec Nodes 25 Edge to node ratio 19 8 to I Show Correlations Within groups Across groups 11 Click Build Network Page 35 of 39 Group Definition MetScape provides the option of creating groups using a group definition file A group definition file is a simple 2 column file with metabolite names in the first column and group names in the second column Group names can be anything that you choose A group definition file can be loaded using the Select button under Group Definition on the MetScape tab If a group definition file is used a Group Filter tab will appear in the Table Panel after the network is drawn Clicking on a group name in the Group Filter tab will select all nodes in the network that are part of that group Correlation Visualization MetScape provides a legend explaining its various shapes and colors The legend will be specific to the current network type pathway or correlation While on a correlation network access the MetScape Legend from Apps gt MetScape gt Show Legend a Legend Known Compound Unknown Compound Y Correlation St
33. rength EE Positive Correlation Negative Correlation 1 Significance Based 5 Cose Page 36 of 39 Additional information about the network is expressed through visualization e Edges o If the dataset only has values O to 1 all edges will be black o Ifa dataset has positive and negative correlation values A pink edge represents a positive correlation A blue edge represents a negative correlation o The thicker the edge the stronger the correlation Note In acorrelation based network selected edges are highlighted yellow instead of red e Nodes o Purple nodes represent mapped compounds o White nodes represent compounds that did not map to a known compound in the MetScape database Additional Information Node and edge attributes are displayed in the Table Panel See the Table Panel Display section above for details on this panel A Pathway Filter tab is also available in the Table Panel See the Pathway Filter in the Table Panel section above for details about this panel When a correlation based pathway is built its name in the Network tab reflects significance information for that network This naming convention is to help distinguish between networks when creating multiple networks in the same Cytoscape session Correlation Network Results Panel Details about nodes can be viewed by double clicking on a node of interest To learn more about the details window for known compounds see the MetScape Re
34. s and node sizes change within the three treatments showing changes over time To stop the animation click Stop on the Animation Controls window Page 28 of 39 Manipulating Network Expanding a network From a compound node you can expand the current network to include additional reactions and related compounds genes and enzymes Option 1 Expanding a network in the current network window Example using Compound Reaction Network 1 Right click on the compound node you want to expand this node is known as the expansion seed node A menu of options will pop up 2 Goto MetScape gt Expand gt Expand in Existing Network X Nested Networks gt Apps ie ROOM a k r MetDisease MetScape Po Create Supetwork M3 U External Links Expand gt Expand in Existing Network i i Collapse Preferences Expand in Subnetwork Restore Original Network i 3 Additional compounds and reactions are added to the network As a result the network is often redrawn 4 The edges between the original node and the expanded nodes are now blue Owed die Page 29 of 39 Option 2 Expanding a network in a new window Example using Compound Network from Selected Genes 1 Right click on the compound node you want to expand this node is known as the expansion seed node A menu of options will pop up 2 Select MetScape gt Expand gt Expand in Subnetw
35. sults Panel section above Double clicking on an unknown compound will bring up a Results Panel that contains links to HMDB ChemSpider MassBank and METLIN Results Panel Node Edge Details Compound Node Links for m z searches HMDB ChemSpider MassBank METLIN Each of these links will allow you to search based on mass to charge values helping to identify the unknown compound Page 37 of 39 In addition a node attribute file can be imported into Cytoscape with one column for nodes and a second column for mass to charge The mass to charge column needs to have one of the following as the heading m z M Z m z M Z mz or MZ If the mass to charge values are entered for unknown compounds clicking on the HMDB link in the Results Panel will go to the HMDB results for that mass to charge value Please note that the other three links will still go to their search pages SAVING AND REOPENING A SESSION Saving a session To save a Cytoscape session containing one or more MetScape app networks 1 Select File gt Save or Save As from the Cytoscape menu 2 Browse to a location for saving the file 3 Name the file 4 Click Save Reopening a session To reopen a saved session containing MetScape app data 1 Select File gt Open from the Cytoscape menu 2 Navigate to the saved file location 3 Select the file 4 Click Open 5 After Cytoscape reports that the session file was successfully loaded click Close
36. uild Different Network Type cccccccsssccccesseccceeseccceeeseceseeeeceseeeeceeseusecessenecetseees 16 Table Panel DISplay enn ee ener eee ene ne eee eee ee 16 Choosing attributes provided through MetScape ccccccsssccccssseccccesecceeenecceeeuseceseeecessegeceeseees 16 Rearranging attributes in the table panel cccccccccccsssccccesececeenececceeeceeeeeseceseuaeceseeeceeseneeeeeas 19 Pathway filter in TNE table panel seers sscecsiencadausesiexcsacwsiveoretieeacsonaacendaaswcsasesdteateaiabeasaveosendeaduedasoreasnanens 20 Concept filter in the table panel ce ciscicessce er scteaie set cscs haeesceah ccs Seantater eatbatea eves cea nen pseueaciuaeeeebarweetens 21 MetScape Results Panel Additional Node Information ccccccccccesssecceceeeesecceeseeeseceeeeaeeeeeess 24 PANINI OND O DAU ccstrsce cca E cto E neuen O E anew 24 Building an PUIUV AUN ONY cesses eeirsciscee on atsadivceeaceancpend east ciedaiese asda ont vsedscec dandaoehsoniaeeeavismeaineseusmmtesseaeraene 24 Manipulating the animation ZOOMING ccccccccsseccccesseccceesececeenececceueceeeeuseceseenecesseeceeseneeeetas 27 Manipulating the animation bar Chart ccccccccsseccccessccccesececsesececeeecceeeusecesseeceseugeceeteneeeenas 28 Playing the animation oot ce cet corsa es cacti ener tseyeeeteoneeee i eee 28 Manipulating Network Qaaeeetenn eee eee ee ee ee en ne ee ee ee ee eee 29 Expanding a CT
37. y To create a subgraph 1 Select a pathway in the Pathway Filter tab 2 Click the Create Subnetwork button at the top of the Table Panel make sure you are on the Pathway Filter tab 3 A new graph appears in the graph window This is the subnetwork for the selected pathway Concept filter in the table panel The content of the created or loaded concept file is put into the Concept Filter tab Table Panel ay Concept Name 1 4 Dichlorobenzene degradation 21 9 jl k 1 and 2 Methyinaphthalene degradation 25 7 E 10 293 3 Chloroacrylic acid degradation lis 8 l 0 172 Acute myeloid leukemia 53 E i 10 467 Adherens junction 74 12 it 10 042 Adipocytokine signaling pathway 73 12 fr 10 901 Node Table Edge Table Network Table Panay Fiter concept ite o 1 Concept Name official name of the concept 2 Number of Enriched Driving Genes the number of enriched driving genes in the concept 3 Number of Genes in Network the number of input genes that belong to the concept 4 Direction direction of the change Page 21 of 39 5 P value the p value for enrichment depletion 6 False Discovery Rate the FDR estimated using the method of Benjamini and Hochberg When results are sorted by p value a FDR lt 0 05 is interpreted to mean that approximately 5 of the concepts with lesser or equal p value are false positives TO map a concept on the
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